This section provides background information related to the present disclosure which is not necessarily prior art.
Collagen is the main load-bearing structural protein in the extra cellular matrix (ECM) of biological tissue. The quantity, type, and orientation of collagen greatly influences the way tissues carry pressure, stretch, and maintain integrity. For instance, skin wounds transit from weaker type III collagen to type I collagen, improving wound integrity in the later part of healing. Also in the ocular realm, structural composition of type I versus type III collagen varies in different parts of the ocular tissue, which may be linked to diseases such as glaucoma.
Several techniques are currently used to measure tissue collagen compositions, such as histological staining, histological autofluorescence, and non-invasive tissue optical biopsy. Tissue histology provides well-established analysis and quantification methods in techniques such as immunofluorescence and picrosirius staining microscopy. However, histology requires invasive sampling and destructive sectioning to produce samples for measurement.
Alternatively, autofluorescence of collagen can be exploited in various analytical techniques. Autofluorescence from collagen, i.e., without staining, is commonly observed in cell and tissue microscopy. The major collagen fluorophores are lysine derived pyridinium, tyrosine, and phenylalanine groups, which can be affected by crosslinking, glycation, and the overall compositional differences between types of collagens. Collagen autofluorescence, in combination with other endogenous fluorophores, can provide differentiation between normal and cancerous tissues, promising tumor demarcation in minimally invasive surgeries. Moreover, collagen fiber orientation and crystallinity enable second harmonic generation (SHG) using laser induced autofluorescence. Both multiphoton collagen autofluorescence and SHG have been adapted for monitoring skin aging and investigating ocular pathology. Despite the ability of SHG and multiphoton microscopy to detect endogenous fluorophores, e.g., NADH+, elastin, and collagen, no differentiation of collagen types have been demonstrated. This lack of differentiation is because very little differences exist in the spectral domain of collagen types, e.g., I versus III.
However, autofluorescence of different collagen types evidently have strong differences in the time domain—their lifetimes are significantly different and sensitive to the crosslinking and glycation, as mentioned above. Therefore, time domain fluorescence lifetime spectroscopy can provide differentiation of collagen types in tissue. However, laser-induced tissue fluorescence and microscopy techniques have been developed for non-invasive tissue monitoring, but have not enabled differentiation of collagen types specifically. Laser fluorescence and microscopy instrumentation is also prohibitively costly and complicated for widespread biomedical application. Accordingly, there remains a need to develop cost effect and non-invasive methods for measuring tissue collagen compositions.